Keratoconus (KC), with an incidence of ~1/1,000, is a degenerative weakening and thinning of the corneal connective tissue extracellular matrix (ECM), causing high astigmatism, scarring, and vision loss in severe cases. There are no curative treatments: the disorder is managed by hard contact lens use and UV cross-linking of collagens (CXL) to temporarily strengthen the connective tissue, at early stages, and cornea transplants, at advanced stages. However, the long-term effects of CXL are unknown and as KC onsets at a young age, it impacts ones productivity and quality of life tremendously. We hypothesize that dysregulated stress response in keratocytes, the specialized cells of the cornea and disruptions in the stromal ECM they produce are major disease underpinnings. KC is multifactorial with a strong genetic component: 18% of cases have a positive family history, concordance is significantly higher in monozygotic than dizygotic twins, and KC is positively associated with heritable traits like central corneal thickness and genetic disorders like trisomy 21 and Ehlers- Danlos syndrome types. Past linkage, candidate gene sequencing and genome-wide association studies (GWAS) have led to suggestive clues but have not identified causal genes. Consequently, we propose a functionally- complemented exome sequencing study that relies on three major advances from our laboratory: (1) We have performed proteomics and preliminary RNA-Seq screens of KC and control corneas to identify the universe of known and novel corneal RNAs and proteins within which genetic defects occur. (2) We have established a 'disease-in-a-dish' model using keratocytes from individual donor (DN) and KC corneas, where KC cells mimic degenerative features of the cornea. (3) Our expression and functional studies of KC indicate a dysregulated network involving cytokines, TGFb and AKT, as major effectors of cellular stress and ECM perturbations in KC. Aim I will investigate TGFb and PI3K/AKT signal transductions in cellular stress and ECM/collagen disruptions in KC cell cultures. Aim II will identify transcriptional alterations in individual KC and DN corneas by RNA-sequencing. Aim III will identify KC susceptibility genes by exome sequencing of familial and isolated cases. We have established an active Keratoconus Research Center at Johns Hopkins Wilmer Institute for long-term patient recruitment, locally and through national/international collaborators, with an experienced research team in ocular medicine (Walter Stark, Albert Jun), bioinformatics, genomics and proteomics (Steven Salzberg, Aravinda Chakravarti, Akhilesh Pandey) and ECM cell biology and biochemistry (Shukti Chakravarti). Our findings will elucidate keratoconus pathogenesis at a molecular level and lead to strategies for therapeutic interventions.